PROJECT SUMMARY/ABSTRACT Multiple sclerosis (MS) is an inflammatory demyelinating neurologic disorder thought to be caused by immune-mediated injury against the central nervous system (CNS). The adaptive immune system, in particular, is considered to be a central mediator of MS immunopathogenesis. CD8+ T cells are best known for their cytotoxic function in viral and tumor immunity, yet compelling evidence suggests an important, but still largely unknown role in MS. Deciphering which CD8+ T cells are relevant to the disease process and what unique characteristics they possess is a critical hurdle to our understanding of MS. Studies analyzing the phenotype of T cells in the cerebrospinal fluid (CSF) of MS patients have indicated increased levels of pro-inflammatory cytokines and activation status, however, these studies have focused on a relatively narrow range of markers. Transcriptomics, using techniques such as RNA-Seq, provide an avenue for studying a far vaster array of phenotypic markers. Single cell (sc)RNA-Seq is a relatively recently developed technology that allows high throughput gene expression, thereby revealing the molecular profile of cells in exquisite detail. To our knowledge, there have been no published single cell RNA-Seq studies on CD8+ T cells in MS. Although prior studies of the intrathecal T cell receptor (TCR) repertoire have suggested greater CD8+ T cell clonality compared to CD4+ T cells in MS, this has been limited to Vb analysis of bulk T cells. Thus, the true clonal frequency of CD8+ T cells in the CSF is unclear. In addition, the lack of available paired TCR ab sequence data precludes the future identification of T cell antigen specificity using unbiased antigen discovery platforms. Very recently it became possible to combine scRNA-Seq and TCR ab sequencing. The objective of this proposal is to characterize specific CD8+ T cell sub-populations that are most likely contributory to MS by measuring the clonal frequency and transcriptional profile by high throughput single cell sequencing. It is hypothesized that clonally expanded CD8+ T cells from MS patients will have unique gene expression profiles within the CSF compartment where disease-relevant cells are likely enriched. As an extension of this unbiased approach, we will also perform single cell sequencing of newly identified myelin- specific CD8+ T cells in MS patients, in order to determine their frequency and phenotype in the CSF. These studies are expected to yield valuable insights into the features of CD8+ T cells that are likely relevant to MS pathogenesis.